US 12,066,446 B2
Methods for detecting dihydroxyvitamin D metabolites by mass spectrometry
Brett Holmquist, Mission Viejo, CA (US); Nigel Clarke, Vista, CA (US); Anne Caston, San Pedro, CA (US); and Richard E. Reitz, Las Vegas, NV (US)
Assigned to Quest Diagnostics Investments Incorporated, Wilmington, DE (US)
Filed by Quest Diagnostics Investments Incorporated, Wilmington, DE (US)
Filed on May 2, 2023, as Appl. No. 18/142,153.
Application 18/142,153 is a continuation of application No. 16/748,234, filed on Jan. 21, 2020, granted, now 11,650,216, issued on May 16, 2023.
Application 16/748,234 is a continuation of application No. 14/267,014, filed on May 1, 2014, abandoned.
Application 14/267,014 is a continuation of application No. 13/751,915, filed on Jan. 28, 2013, granted, now 8,852,951, issued on Oct. 7, 2014.
Application 13/751,915 is a continuation of application No. 13/436,651, filed on Mar. 30, 2012, granted, now 8,389,292, issued on Mar. 5, 2013.
Application 13/436,651 is a continuation of application No. 13/117,997, filed on May 27, 2011, granted, now 8,173,442, issued on May 8, 2012.
Application 13/117,997 is a continuation of application No. 11/946,765, filed on Nov. 28, 2007, granted, now 7,972,868, issued on Jul. 5, 2011.
Prior Publication US 2023/0266347 A1, Aug. 24, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. G01N 33/82 (2006.01)
CPC G01N 33/82 (2013.01) [Y10T 436/10 (2015.01); Y10T 436/20 (2015.01); Y10T 436/203332 (2015.01); Y10T 436/24 (2015.01)] 7 Claims
 
1. A method for determining an amount of 1,25-dihydroxyvitamin D in a serum sample by tandem mass spectrometry; the method comprising:
(i) adding a deuterated internal standard to a serum sample;
(ii) precipitating protein from the sample with acetonitrile;
(iii) purifying the 1,25-dihydroxyvitamin D and the deuterated internal standard by solid phase extraction (SPE);
(iv) derivatizing the 1,25-dihydroxyvitamin D and the deuterated internal standard with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) to form a derivatized 1,25-dihydroxyvitamin D and a derivatized deuterated internal standard;
(v) subjecting the derivatized 1,25-dihydroxyvitamin D and the derivatized deuterated internal standard to liquid chromatography;
(vi) ionizing the derivatized 1,25-dihydroxyvitamin D and the derivatized deuterated internal standard to produce one or more 1,25-dihydroxyvitamin D precursor ions having a mass/charge ratio of 586.37±0.5 and one or more the deuterated internal standard precursor ions having a mass/charge ratio of 314±0.5;
(vii) fragmenting the one or more 1,25-dihydroxyvitamin D precursor ions and the internal standard precursor ions into one or more 1,25-dihydroxyvitamin D precursor fragment ions and/or deuterated internal standard precursor fragment ions detectable by mass spectrometry;
(viii) determining an amount of the one or more 1,25-dihydroxyvitamin D fragment ions and the one or more deuterated internal standard fragment ions; and
relating the amount of the one or more 1,25-dihydroxyvitamin D fragment ions to the amount of 1,25-dihydroxyvitamin D in the serum sample.